Leukocyte inactivation module

ABSTRACT

The present invention provides a module for reducing the activity of leukocytes which comprises a carrier and a ligand that is linked to the carrier and is suitable for interacting with a leukocyte receptor. Furthermore, the present invention provides a process for reducing the activity of leukocytes using said module. The advantage of the present invention is that after binding the activated leukocytes in the leukocyte inactivaton module (LIM), the damaging effect of the cells is inhibited within minutes.

FIELD OF THE INVENTION

[0001] The present invention relates to a leukocyte inactivation module(LIM) and a process for reducing the activity of leukocytes.

BACKGROUND OF THE INVENTION

[0002] An acutely increased pathologic cellular immune responsefrequently occurs in various clinical situations. Examples therefor are:

[0003] The arterial or venous line of a heart-lung-machine duringcardiosurgery;

[0004] Systemic Immune Response Syndrome (SIRS) after multiple injuriesor sepsis (septic shock);

[0005] Multiple Organ Dysfunction Syndrome (MODS) due to excessiveimmune activity.

[0006] During the above-mentioned surgeries or clinical complications,an undesired activation of leukocytes occurs resulting in severepathological complications in the patient. To avoid this, the activatedleukocytes (in particular, neutrophils) should be removed from the bloodstream and inactivated immediately. In the currently available leukocytefilters, an increased number of activated leukocytes are filtered offfrom the blood. However, the cells still living produce and secretepathogenic substances (cytokines, enzymes, oxygen radicals etc.) thatare responsible for the actual pathogenesis. There are indications thatthe leukocytes in the filter net are additionally activated presumablythrough mechanical stress und through the contact of the cells with theforeign surface. As an example, the enzyme elastase produced byactivated neutrophils is secreted in a higher amount. Elastase actsinter alia on the extracellular matrix of the vessel wall and cleavesinterendothelial cell-cell contacts resulting in an increasedpermeability of the vessel walls, in edema, in enhanced inflammation andthe like.

[0007] Apoptosis is one of the most important regulation elements of theimmune system. The apoptosis of immunrelevant cells results in anormalisation of the activity of the immune system after an immuneresponse, e.g. against microbial pathogens. Also during the individualdevelopment, the immune system must kill those immune cells acting onendogenous structures or on natural antigens from the environment (e.g.autoimmune diseases or allergies). T-cells being activated via so-calledantigen-presenting cells (apc) usually receive several pieces ofinformation. The antigen processed by the apc is presented in the groupof the MHC-I or MHC-II molecule. If the affinity of the T-cell receptorto the antigen is too weak or in the absence of co-stimulating signals(e.g. via adhesion molecules), the cell becomes apoptotic. Anotheressential mechanism resulting in apoptosis is started via the Fas/FasLpathway. In this mechanism, e.g. endothelial cells of the vessel wallsor other epithelial cells can express FasL thus protecting the tissuefrom entering activated immune cells.

[0008] Problem to be Solved by the Invention

[0009] It is the aim of the present invention to provide a devicesuitable for reducing the activity of leukocytes, thereby reducing thesecretion of pathogenic substances by the leukocytes. Furthermore, thepresent invention is to provide a process for reducing the activity ofleukocytes using such device.

[0010] Means for Solving the Problem

[0011] The present inventors carried out investigations withcytomegalovirus-infected retinal pigment epithelial cells from the humaneye and found that, due to the contact with FasL on the epithelialcells, activated neutrophils lost their ability to maintain or increasethe adhesion to the epithelial cells. This surprising result is supposedto be a protecting mechanism of the endothelium and the respectivetissue against inflammatory incidents. The functional loss of theneutrophil-effector-mechanisms could be observed within minutes aftercell-cell contact and seems to be largely independent of the apoptoticsignal pathway in the neutrophils. On the basis of these surprisingfindings, e.g. the Fas/FasL pathway or other early inhibitory mechanismsof the leukocyte-effector functions can be employed for the experimentaland clinical use for acute excessive immune reactions.

[0012] Subject-Matter of the Invention

[0013] The present invention provides a module for reducing the activityof leukocytes, which comprises a carrier and a ligand that is linked tothe carrier and is suitable for interacting with a leukocyte receptor.Furthermore, the present invention provides a process for reducing theactivity of leukocytes using said module.

[0014] The advantage of the present invention is that after binding theactivated leukocytes in the leukocyte inactivation module (LIM), thedamaging activity of the cells is inhibited within minutes. This is dueto the contact of specific receptors on the cell membrane of theleukocytes with the respective ligands in the LIM. The ligands can beproteins inducing, after contact with the receptor on the cell membrane,a signal that stimulates leukocytes to reduce the secretory activity andthe immunogenicity. A possibility to achieve this is the induction ofapoptosis via relevant receptor-ligand interactions, e.g. Fas/FasL.

DESCRIPTION OF THE INVENTION

[0015] The module according to the present invention is suitable forbeing introduced into the patient's blood stream using a Shaldoncatheter or into the circulation of a heart-lung machine.

[0016] The module preferably consists of a plastic housing with adiameter of e.g. 10 cm. The blood inlet nozzle and the blood outletnozzle are adapted to the tube connections of the heart-lung machine.There is a carrier in the module, e.g. a three dimensionally foldedpolyester membrane with modified surface for the adhesion of activatedleukocytes and for their inactivation and killing (e.g induction ofapoptosis) via receptor-induced signals. The carrier material can be anymaterial that is suitable for binding ligands. The term “binding” usedherein comprises both covalent and non-covalent binding, e.g. saltbinding, hydrophobic interactions and affinity binding, of a ligand tothe carrier. Furthermore, the ligand may be bound directly or indirectlyto the carrier. The indirect binding comprises binding via a bindingmediator, e.g. a long-chain molecule, for a better presentation of theligand or via a cell comprising the ligand and being bound to thecarrier via another binding interaction.

[0017] The LIM according to the present invention is suitable for anyleukocytes, i.e. for B-lymphocytes, T-lymphocytes, granulocytes,neutrophils.

[0018] The other parameters of the module determining the blood stream,pressure or rheology are the same as in conventional leukocyte filtersthat are already used clinically.

EXAMPLE 1

[0019] The wells of a 12-well culture plate were lined with polyestermembranes (pore size of 40 μm) and incubated overnight with variousconcentrations of a functionally activated IgM antibody against Fas(CD95). The following controls were used:

[0020] wells without membrane

[0021] wells with membrane, no pre-incubation with antibody was carriedout

[0022] wells with membrane, pre-incubation with irrelevant IgMantibodies was carried out.

[0023] Fas-expressing (Fas+) and Fas-deleted (Fas−; expresses no Fas onthe surface) Jurkat cells as test cells were added to the wells in aconcentration of 1×10⁶/ml for 24 hours.

[0024] The apoptosis rate and the necrosis rate were determinedquantitatively by flow cytometry using an annexin binding assay.

[0025] Results: The Fas-Jurkat cells showed no significant increase inthe annexin V binding property after cultivation in the pre-treatedwells. In contrast thereto, a significant induction of apoptosis showedin dependency on the concentration of the IgM antibody. Fas+ Fas−  0 ng24.81% 24.29% (control value)  10 ng 31.38% 27.57%  50 ng 40.95% 26.20%100 ng 89.31% 29.65%

[0026] In the above controls, no induction of apoptosis could be found.Similar results were obtained with freshly isolated neutrophils.

[0027]FIG. 1 shows the results of the example of the present invention.

1. A module for reducing the activity of leukocytes, which comprises acarrier and a ligand that is linked to the carrier and is suitable forinteracting with a leukocyte receptor.
 2. The module according to claim1, wherein the ligand is a protein that is suitable for interactingspecifically with the receptor.
 3. The module according to claim 1 or 2,wherein the ligand is linked to a binding mediator that is linked to thecarrier.
 4. The module according to claim 3, wherein the bindingmediator is a cell.
 5. The module according to claim 4, wherein thebinding mediator is a cell and the ligand is the FasL protein.
 6. Themodule according to any one of claims 1 to 4 wherein the ligand is anantibody against the leukocyte receptor.
 7. The module according toclaim 6, wherein the ligand is an antibody against the Fas protein ofleukocytes.
 8. A process for reducing the activity of leukocytes bycontacting with ligands in a module according to any one of claims 1 to7.
 9. The process according to claim 8, wherein the leukocytes, aftercontacting with the ligands, secrete cytokines and/or enzymes and/oroxygen radicals in lower amount than before contacting.
 10. The processaccording to claim 8 or 9, wherein the interaction of the ligand linkedto a cell with the leukocyte receptor reduces the activity of theleukocytes in terms of binding affinity to the cell.